The present invention relates to a method of producing human papillomavirus (HPV) pseudovirions in plant cells, the plant produced pseudovirions per se, a neutralisation assay using the plant produced pseudovirions and pharmaceutical compositions comprising the plant produced pseudovirions.
The applicants have use novel autonomously replicating vectors in conjunction with previously developed, non-replicating vectors to produce HPV-16 pseudovirions (PsVs) in plants. Preliminary expression trials established optimal conditions and timeframes for production of each individual element required for assembly of HPV PsVs. The structural elements required for PsV production are HPV L1 and L2 proteins, produced by non-replicating plant expression vectors pTRAc-hL1 and pTRAc-hL2, respectively; and circular double-stranded DNA from one of three replicons derived from pRIC3-mSEAP, pRIC3-mSEAP+ or pRIC3-mluc+. Putative PsV particles, as well as L1/L2 virus-like particles (VLPs) produced in absence of the replicons, were harvested from plants and purified by successive gradient ultracentrifugation steps. Gradient fractions containing L1 were pooled and dialysed against high-salt (0.5M) NaCl PBS, to obtain purified PsVs. These were confirmed by electron microscopy to be conformationally similar to VLPs and PsVs produced in other systems, and by PCR to contain the corresponding encapsidated replicon DNA. Purified PsVs were used to demonstrate their use in a neutralisation assay. Two of the three PsVs created, namely mSEAP and mluc+ PsVs, demonstrated successful pseudoinfection and neutralisation with a common HPV16 neutralising antibody, while mSEAP+ PsVs showed no reporter gene expression after pseudoinfection of mammalian cells. This is the first known report of the production and purification of HPV PsVs, as well as L1/L2 VLPs in planta, as well as the first demonstration of a pseudovirion-based neutralisation assay (PBNA) using plant-produced PsVs.
Cervical cancers caused by high-risk HPV are the second most prevalent form of cancer in women in developing countries. Africa in particular has been identified as a high risk region for the disease. Recently developed L1 VLP vaccines, Cervarix® and Gardasil®, protect against HPV-16 and HPV-18, or HPV-6, HPV-11, HPV-16 and HPV-18 infection, respectively. Both currently available vaccines, Cervarix® and Gardasil®, elicit a strong and protracted neutralising antibody response, and have been shown to have sustained efficacy up to 5 years post-administration. While these vaccines have shown great promise in reducing the burden of disease, development and production of VLP vaccines remains prohibitively expensive, particularly in developing countries.
A key element of any HPV vaccine development initiative is the pseudovirion-based neutralisation assay (PBNA). Induction of neutralising antibodies is currently the best estimate of vaccine candidate efficacy for second generation HPV vaccine testing. Until recently, the identification of serum neutralising antibodies relied on the use of enzyme-linked immunosorbent assay (ELISA) or neutralisation assays using whole virus (Dessy et al., 2008). However, improvements in HPV PsV production efficiency in the last decade have allowed the development of the PBNA. Developed by John Schiller's group at the Center for Cancer Research, this assay uses mammalian cells for intracellular production of PsVs expressing a secreted alkaline phosphatase (SEAP) reporter gene (Buck et al., 2005a), and has since become the gold standard for testing neutralisation of candidate HPV vaccines, allowing rapid and un-biased screening of neutralising antibodies and epitopes (Stanley et al., 2008). While this production method has been shown to be extremely effective for production of PsVs, cell culture production is expensive, and SEAP assay kits are particularly expensive in comparison to other commonly used reporter assays such as luciferase or GFP. There is a need, therefore, to develop alternative PsV production methods to allow for affordable candidate vaccine development and, in particular, inexpensive testing of immune sera.
The production of neutralising IgG antibodies in response to vaccination has long been understood to be a key aspect of protective immunity (Robbins et al., 1995). It has been suggested that it may be possible to accurately estimate the required level of neutralising antibody required for protection, provided that the concentration, isotype and secondary biological activity of these antibodies could be accurately measured (Robbins et al., 1995). Neutralisation assays were developed as a method of accurately quantifying the neutralising capabilities of immune sera, usually in response to a live viral or vaccine candidate challenge, as well as identify neutralising epitopes (Ochsenbauer and Kappes, 2009; Yeager et al., 2000).
The first demonstration of in vitro neutralisation of papillomavirus was by Dvoretzky et al. (1980), who demonstrated neutralisation with rabbit-produced Bovine papillomavirus type 1 (BPV-1) antisera to confirm the role of BPV-1 in focus formation in mouse cell lines. Early efforts to establish a robust, sensitive in vitro neutralisation assay for HPVs were hampered by difficulties in production of infectious virus. Production of infectious virions in vitro was first achieved by grafting HPV-11-infected material into athymic mice—grafts were left to develop into condylomatous cysts over a period of 3-5 months, before being harvested and purified for HPV virions (Kreider et al., 1987). This method was utilised to produce virions for use in the first de facto neutralisation assay. Neutralising monoclonal antibodies were identified and isolated from HPV-11 or BPV-1 antisera. These antibodies were then used to demonstrate neutralisation of intact virions by ELISA, as well as identifying several neutralising conformational epitopes (Christensen et al., 1990). The same group used this method to successfully identify neutralising HPV antibodies in human sera for the first time, and further demonstrated that ELISA was a good indicator of the presence of neutralising antibodies in human sera (Christensen at al., 1992). Another approach coupled the neutralisation of HPV-11 infection with RT-PCR detection of HPV mRNA transcripts to create a semi-quantitative neutralisation assay (Smith et al., 1995). While these approaches were nominally successful in identification of neutralising antibodies, detection remained limited at best, and the procedures used were time-consuming and expensive.
A major step forward in neutralisation assay technology came with the advent of PsV production, which abrogated the need for the expensive and time-consuming xenograft production method. Roden at al. (1996) used hamster BPHE-1 cells to generate BPV-1 or HPV-16 PsVs. These were used to demonstrate focus formation in C127 cells, using the technique demonstrated by Dvoretzky et al. (1980). These researchers further showed that neutralising antibodies in HPV-16 antisera prevented focus formation, demonstrating a quantitative neutralisation assay of a high-risk HPV type using PsVs for the first time (Roden et al., 1996). In this report, the authors noted that the focus transformation assay required 2-3 weeks, and that inclusion of a marker or reporter gene would greatly improve the speed of the assay. This was first attempted by chemically linking a β-lactamase (BLAM) reporter plasmid to VLPs or infectious virions, and incubating these with PV antisera before infecting various mammalian cell lines. Early attempts demonstrated neutralisation, but resulted in <1% infection of cells with these PsVs (Muller at al., 1995). Yeager at al. (2000) and Bousarghin et al. (2002) demonstrated this approach more successfully, using a BLAM or luc reporter plasmids and an alternative method of attaching the plasmid to VLPs. More importantly, several groups generated PsVs with encapsidated reporter genes, and demonstrated their use for neutralisation assays (Buck et al., 2005a; Fleury et al., 2008; Kawana at al., 1998; Rossi at al., 2000; Stauffer at al., 1998; Touze and Coursaget, 1998; Unckell et al., 1997). While early attempts were inefficient due to poor PsV production levels, this was improved upon by intracellular generation of high yields of L1/L2 PsVs and Incorporation of a SEAP reporter plasmid (Buck at al., 2004). These PsVs were used with a commercially available SEAP detection kit to demonstrate a pseudovirion-based neutralisation assay that was at least as sensitive as, and potentially more type-specific than, the standard ELISA-based neutralisation assay (Pastrana et al., 2004).
While the system developed by Pastrana et al. (2004) is considered the current ‘best practice’ neutralisation assay, there remains room for improvement. In particular, the costs of PsV production could be greatly decreased by the use of a less expensive production system (Brondyk, 2009). Recombinant protein expression in plants has been demonstrated to have a significantly lower cost of production when compared to production in mammalian cells (Tiwari et al., 2009). As such, plant expression may provide an attractive alternative for the production of PsVs for use in the PBNA.
Expression of recombinant proteins in plants has developed over the last twenty years from a curiosity in the late 1980s to a medically and industrially relevant production system today. Early efforts relied on transformation of plants to produce stable transgenic lines. This was achieved through biolistic delivery or, more recently, agroinfiltration (Daniell et al., 2009). While transgenic protein production remains a useful and viable system, advances in transient expression methods and technology have positioned transient expression as the preferred method for industrial-scale production in plants (Rybicki, 2010). Two key factors that have played a central role in this transition are viral, or virus-derived, expression vectors, and the development of agroinfiltration technology.
Agroinfiltration was originally developed to as an alternative to biolistic bombardment for the stable transformation of plants (Kapila et al., 1997). This process relies on the DNA transfer capability of A. tumefaciens to introduce foreign DNA to plant cells. A. tumefaciens can be used to transfer a transgene located in the transfer DNA (T-DNA) segment of the Ti plasmid into plants infiltrated with a bacterial suspension of the transformed bacterium. The T-DNA is transported to the plant nucleus, and this allows for transformation of the plant through integration of the T-DNA into the plant genome (Zupan et al., 2000). Importantly, however, a transgene incorporated into the T-DNA may also be transiently expressed, from non-integrated or episomal T-DNA, resulting in systemic expression of a recombinant protein without the need for stable transformation (Kapila at al., 1997).
Viral vectors were the first transient expression method developed for plants. Early efforts simply inserted a recombinant gene or epitope into the genome of viruses such as TMV, cowpea mosaic virus (CPMV), or PVX, either fused to the viral coat protein or separately, under control of a duplicated subgenomic viral promoter (Durrani et al., 1998; Gleba et al., 2007; Turpen at al., 1995). While this application produced immunogenic protein, expression levels were lower than those found in transgenic plants. Other problems with these ‘first-generation’ viral vectors included a tendency to revert to the natural virus, constraints on insert size, difficulty of administration, and an inability to form VLPs (Kohl et al., 2006; Rybicki, 2010; Varsani at al., 2006).
These limitations prompted further work to develop ‘second generation’, or deconstructed, viral vectors. This approach used only the desirable viral elements, in particular the replicative machinery, to manufacture synthetic vectors capable of inducing transgene expression in plants. While these vectors are usually not infectious on their own, when coupled with agroinfiltration technology they can result in systemic transient expression of protein at levels comparable to that of transgenic plants (Tiwari at al., 2009). This approach has the advantages of short time frames (3-7 days) when compared to stable transformation (6-9 months), significant expression levels, and rapid and easy scale-up and purification. This makes agroinfiltration-mediated transient expression via viral vectors an ideal approach for the production of medically relevant proteins and particles in plants. Of particular interest is the use of transient expression for the production of VLPs and PsVs in plants, as there is potential for a reduction in cost when compared to traditional systems (Santi et al., 2006).
Papillomavirus L1 VLPs have been produced by several groups in plants. Most have used transgenic plants (Biemelt et al., 2003; Warzecha at al., 2003) with resulting low yields. Early attempts at transient expression of L1 also yielded low levels of expression, as well as an apparent inability to form VLPs (Varsani et al., 2006). However, agroinfiltration of an Agrobacterium vector coding for a human codon-optimised L1 protein provided a much higher protein yield, and demonstrated that transient expression of HPV-16 VLPs at high levels is a feasible approach for the production of immunogenic HPV candidate vaccines (Maclean et al., 2007).
The vector used to produce L1 at such high expression levels—pTRAc—was developed at the Fraunhofer Institute for Molecular Biology and Applied Biology. This vector utilises a CaMV 35S promoter with duplicated transcriptional enhancer, chalcone synthase 5′-untranslated region, and CaMV 35S polyadenylation signal for foreign gene expression. This vector has also been used to express minor capsid protein L2 in plants (Pereira, 2008). However, coexpression of L1 and L2 has not previously been conclusively demonstrated to form VLPs in planta.
A further development in vector technology has been the use of single-stranded DNA plant geminiviruses in the genus Mastrevirus, family Geminiviridae, to create replicating vectors. These replicating vectors incorporate a viral Ori (origin of replication) sequence that is duplicated on either side of a gene expression cassette. The replicating vectors further may or may not include a viral replication-associated protein (Rep) gene. Agroinfiltration of a single Rep-containing replicon construct, or of a replicon construct plus a Rep construct expressed in trans by standard techniques, results in release of a plasmid-like “replicon” which multiplies under the control of Rep protein up to copy numbers of several thousand per cell (Regnard et al., 2010). This can result in significantly increased expression of genes of interest compared to non-replicating vector expression. While the expression of a geminivirus Rep gene and cognate (eg: Ori sequence from the same virus) replicon construct in a plant cell leads to replication of the replicon, this is not known to occur in mammalian cells.
Encapsidation or covalent attachment of DNA by HPV VLPs to form PsVs has been demonstrated in yeast, insect, bacterial and mammalian cell systems (Buck at al., 2005a; Roden et al., 1996; Rossi et al., 2000; Unckell et al., 1997). Buck et al. (2005a) demonstrated that intracellular encapsidation of the pseudogenome is more efficient than in vitro disassembly-reassembly methods for the production of HPV PsVs, probably due to cellular factors that assist in correct assembly of the virions (Buck et al., 2008; Fleury et al., 2008; Peng et al., 2011). Currently, HPV pseudovirions have not been successfully expressed in plant expression systems. As discussed above, transient expression in plants offers several significant advantages for this application: protein expression in plants has been shown to be safe, cheaper than other expression systems, and potentially extremely rapid (Ma at al., 2005; Schillberg et al., 2005). A further significant advantage is that there is no need for downstream processing of proteins (e.g. glycosylation), as for bacterial recombinant protein expression systems (Giorgi et al., 2010). While it has been noted that N-glycosylation may differ in plants (specifically, plants cannot synthesise β-1,4-galactose and sialic acid), this problem can be overcome by recent advances in transgenic tobacco to provide ‘humanised’ glycosylation machinery (Bakker et al., 2006; Gleba et al., 2007). Further, it has been suggested that glycosylated L1 or L2 are not an important part of the assembled virion (Zhou et al., 1993).
In this application the inventors evaluated the feasibility of expressing HPV L1/L2 pseudovirions with an encapsidated mammalian reporter cassette, derived from a replicating geminivirus-derived vector, in planta. To achieve this, pTRAc plasmids expressing L1 and L2 proteins were co-infiltrated into plants with novel autonomously replicating plasmids, developed in this study, to create HPV L1/L2 PsVs. Further, we purified these particles by density-based centrifugation, for subsequent testing in a mammalian system.
This invention describes, for the first time, the successful production of HPV PsVs in plants, and testing of the PsVs in a standard PBNA. HPV L1/L2 VLPs, as well as PsVs containing a mammalian reporter cassette pseudogenome derived from the geminivirus Bean yellow dwarf virus (BeYDV), were produced in large quantities in planta. The particles readily encapsidated the pseudogenome DNA provided by the replicating vectors. Further, they were easily purified, stable at high temperature, and were conformationally indistinguishable from PsVs produced in other systems. Most importantly, they were successfully used to perform a PBNA in mammalian cells. Transient plant-based production of HPV PsVs is a feasible strategy, and should be further investigated as a low-cost alternative to mammalian cell culture for PsV production.